Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699

Abstract

Rifamycin production in A. mediterranei is governed by a single gene cluster consisting of structural, resistance and export, and regulatory genes. The genes characterized here could be modified to produce novel forms of the rifamycins that may be effective against rifamycin-resistant microorganisms.

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of mC7N units in ansamycin and mitomycin antibiotics: a review

“…First of these was the rifamycin biosynthetic gene cluster, which we cloned in collaboration with the group of Hutchinson. 49 The 34 gene cluster consists of genes encoding five type I modular polyketide synthases associated with an amide synthase (rifA-F) and a subcluster of AHBA biosynthesis genes (rifG-I, K-N) as well as, separate from it, rifJ, in addition to genes controlling the postpolyketide synthase elaboration of the rifamycin structures, as well as regulatory expression and product export. Some of the genes in the AHBA subcluster ( Figure 6) and their encoded products were expected based on the proposed pathway, such as rifG (aminoDHQ synthase), rifH (aminoDAHP synthase), rifJ (aminoDHQ dehydratase), rifK (AHBA synthase), but three of the genes were totally unexpected, rifL, rifM and rifN with homologies to oxidoreductases, phosphatases and kinases, respectively.…”

“…Another unexpected gene in the subcluster, rifI, is homologous to DHQ dehydrogenases and its role has so far remained obscure. 49 The role of these genes and their products in AHBA formation was then probed by the construction of an expression cassette containing the rifG-N genes under the control of the actII-orf4 promoter and its expression in Streptomyces coelicolor. 50 Efficient AHBA production (350-400 mg l À1 ) by the transformant showed that these genes are sufficient for AHBA formation in this heterologous background.…”

“…50 Lack of an alternative candidate and several pieces of indirect evidence led us to consider the possibility that the RifK protein, in addition to its role as AHBA synthase, may have a second function as the transaminase recruiting nitrogen to the oxidized UDP-glucose to give UDP-kanosamine. Pointing in that direction are (1) the fact that RifK is more closely related to various glutamine-dependent aminotransferases operating in aminoglycoside biosynthesis, such as StsC, than to PLP-dependent enzymes catalyzing a,b eliminations, 49 (2) the close physical association of the AHBA synthase gene with rifL and rifM homologs in all the AHBA synthesis subclusters, suggesting that they may represent a transcription unit that has been recruited intact by lateral gene transfer, (3) the requirement for two separate AHBA synthase genes in ansamitocin biosynthesis, one associated with rifL and rifM homologs and the other with downstream AHBA synthesis genes and (4) the fact that rifK mutants do not accumulate aminoDHS or its spontaneous aromatization product, protocatechuic acid, and that despite an extensive search no other accumulation product could be found. 50 The presumed function of rifM as the enzyme converting UDPkanosamine into kanosamine has not yet been demonstrated, although the protein has been expressed in soluble form.…”

Synthetic Biology in Action: Developing a Drug Against MDR-TB

“…Also, other methods of modification are generally not applicable to this molecule or the producer organism. However, with the availability of rifamycin polyketide synthase gene cluster (rifPKS) in 1998 [12] (Fig. 3) and with the development of cloning vectors and transformation system for the rifamycin producer strain A. mediterranei by our group here at the University of Delhi [13][14][15][16][17][18], the possibilities of manipulating the rifPKS to produce more effective analogues of rifamycin B were raised [16].…”

“…Even though, the co-linear nature of the rifamycin polyketide synthase gene cluster was decrypted in 1998 [12], still, genetically modifying it seemed to be a daunting task. However, by exploiting the modular nature of rifPKS biosynthetic gene cluster, we for the first time could manipulate and modify the rifamycin polyketide ansa chain by combinatorial biosynthetic approach.…”